mutation of a c-terminal motif affects kaposi's sarcoma-associated

JOURNAL OF VIROLOGY, Aug. 2011, p. 7881–7891 Vol. 85, No. 150022-538X/11/$12.00 doi:10.1128/JVI.00138-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Mutation of a C-Terminal Motif Affects Kaposi’s Sarcoma-AssociatedHerpesvirus ORF57 RNA Binding, Nuclear

Trafficking, and Multimerization�

Adam Taylor,1 Brian R. Jackson,1 Marko Noerenberg,1 David J. Hughes,1 James R. Boyne,1†Mark Verow,1 Mark Harris,1,2 and Adrian Whitehouse1,2*

Institute of Molecular and Cellular Biology, Faculty of Biological Sciences,1 and Astbury Centre for Structural Molecular Biology,2

University of Leeds, Leeds LS2 9JT, United Kingdom

Received 20 January 2011/Accepted 3 May 2011

The Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF57 protein is essential for virus lytic replication.ORF57 regulates virus gene expression at multiple levels, enhancing transcription, stability, nuclear export,and translation of viral transcripts. To enhance the nuclear export of viral intronless transcripts, ORF57 (i)binds viral intronless mRNAs, (ii) shuttles between the nucleus, nucleolus, and the cytoplasm, and (iii)interacts with multiple cellular nuclear export proteins to access the TAP-mediated nuclear export pathway.We investigated the implications on the subcellular trafficking, cellular nuclear export factor recruitment, andultimately nuclear mRNA export of an ORF57 protein unable to bind RNA. We observed that mutation of acarboxy-terminal RGG motif, which prevents RNA binding, affects the subcellular localization and nucleartrafficking of the ORF57 protein, suggesting that it forms subnuclear aggregates. Further analysis of themutant shows that although it still retains the ability to interact with cellular nuclear export proteins, it isunable to export viral intronless mRNAs from the nucleus. Moreover, computational molecular modeling andbiochemical studies suggest that, unlike the wild-type protein, this mutant is unable to self-associate. There-fore, these results suggest the mutation of a carboxy-terminal RGG motif affects ORF57 RNA binding, nucleartrafficking, and multimerization.

Kaposi’s sarcoma-associated herpesvirus (KSHV) or humanherpesvirus 8 is a gamma-2 herpesvirus associated with multi-ple AIDS-related malignancies, including Kaposi’s sarcoma,primary effusion lymphoma, and multicentric Castleman’s dis-ease (10, 11, 19). Like all herpesviruses, KSHV has two distinctphases within its life cycle: latent persistence and lytic reacti-vation. During latency the virus expresses a limited subset ofgenes ensuring the persistence of the genome as a high-copy-number nonintegrated episome but remains undetected by thehost immune defense mechanisms (16, 27, 40). After reactiva-tion the virus enters lytic replication, which is typified by acascade of viral gene expression, culminating in the productionof new infectious virus particles (17, 50).

However, in contrast to other oncogenic herpesviruses,where latent gene expression plays a prominent role in tumor-igenesis, lytic replication also plays an important part in thetumorigenicity, pathogenesis, and spread of KSHV infection(19). Specifically, lytic replication appears to be a necessaryantecedent step in KS development from the primary target ofviral infection, the B lymphocyte reservoir, to endothelial cellswhere tumors are observed. Moreover, lytic gene expressionpotentially contributes to the development of KS through the

expression of lytic viral proteins which mediate paracrine se-cretion of growth and angiogenic factors that are essential fortumor growth and development (19). In addition, they sustainthe population of latently infected cells that would otherwisebe reduced due to the poor persistence of the KSHV episomeduring spindle cell division (24).

Posttranscriptional events that regulate mRNA biogenesisare fundamental to the control of gene expression (37). As aconsequence, cells have evolved a “gene expression productionline” that encompasses the routing of a nascent transcriptthrough multimeric mRNA-protein complexes that mediate itssplicing, polyadenylation, nuclear export, and translation (38).These pathways are particularly important for herpesviruseswhich replicate in the host-cell nucleus and express numerouslytic intronless mRNAs (6). Due to the reliance of herpesvi-ruses on the host cell machinery for efficient processing of theirmRNAs, an immediate issue arises concerning the mechanismby which the viral intronless mRNAs are efficiently exportedfrom the nucleus, given that the majority of cellular bulkmRNA nuclear export is intimately linked, and dependentupon, splicing (32, 49).

To circumvent the problem associated with efficient intron-less viral mRNA nuclear export, KSHV encodes a conservedmultifunctional protein, ORF57, which enhances this nuclearexport and the translation of viral intronless transcripts (3, 4,35). To this end, KSHV ORF57 binds viral intronless mRNAs,shuttles between the nucleus, nucleolus, and the cytoplasm,and mediates the nuclear export of intronless viral mRNAs viaa TAP-mediated pathway (3, 5, 7). These properties are alsoconserved in ORF57 homologues throughout herpesvirusessuch as ICP27 from herpes simplex virus type 1 (HSV-1) (12,

* Corresponding author. Mailing address: Institute of Molecularand Cellular Biology, Astbury Centre for Structural Molecular Biol-ogy, University of Leeds, Leeds LS2 9JT, United Kingdom. Phone: 44(0)113 3437096. Fax: 44 (0)113 3435638. E-mail: [email protected].

† Present address: Division of Biomedical Sciences, School ofLife Sciences, University of Bradford, Bradford BD7 1DP, UnitedKingdom.

� Published ahead of print on 18 May 2011.

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13, 28, 43), SM protein from Epstein-Barr virus (EBV) (41,45), and herpesvirus saimiri (HVS) ORF57 protein (2, 8, 14,15, 22).

To gain access to the TAP-mediated nuclear export path-way, ORF57 orchestrates the assembly of an export competentviral ribonucleoprotein particle (3). ORF57 specifically re-cruits the cellular hTREX complex to intronless viral mRNAvia a direct interaction with the hTREX protein Aly, which inturn recruits the cellular protein TAP, which in turn leads tothe TAP-mediated nuclear export pathway (3). However, re-sults suggest redundancy exists in the eukaryotic system forcertain hTREX components involved in the mRNA nuclearexport of intronless viral mRNAs, since experiments involvingsmall interfering RNA-mediated depletion of Aly report only alimited effect on ORF57-mediated KSHV intronless mRNAexport (34). These data correlate with depletion-related stud-ies on the role of Aly in mRNA export in higher eukaryoteswhere, surprisingly, Aly has been shown to be dispensable inmRNA export (20, 31). Therefore, other cellular mRNAadapter proteins, such as UIF (26), RBM15, and OTT3 (48),which interact with TAP are likely to function in ORF57-mediated nuclear export.

Mutational analyses of the ORF57 sequence have revealedseveral functional motifs that are required for RNA binding,nuclear localization, nucleolar trafficking, and multimerization(33). ORF57 contains several putative RNA-binding domains,including an amino-terminal arginine-rich region that resem-bles RNA-binding motifs (ARMs) described in other membersof the family, including EBV SM, cytomegalovirus (CMV)UL69, and HVS ORF57. ORF57 also contains two separatearginine-glycine-glycine (RGG) motifs, similar to an RGG boxmotif, responsible for RNA binding in HSV ICP27 (36). Nu-clear targeting is mediated by three functionally independentnuclear localization signals (NLS) (34), and NLS2 or NLS3 incombination with NLS1 are able to function as a nucleolartargeting signal (7). The carboxy-terminal contains a potentialleucine zipper motif, a zinc finger-like motif. and a GLFFmotif, conserved in gamma herpesvirus ORF57 homologues(21); however, it remains unknown whether these domains areimportant for ORF57 function.

We investigated here the implications on subcellular traf-ficking, hTREX complex recruitment, and ultimately nuclearmRNA export of an ORF57 protein unable to bind RNA. Weobserved that mutation of a carboxy-terminal RGG motif,which prevents RNA binding, affects the subcellular localiza-tion and nuclear trafficking of the ORF57 protein, suggesting itforms subnuclear aggregates. Further analysis of the mutantshows that although it still retains the ability to interact withcellular nuclear export proteins, it is unable to export viralintronless mRNAs from the nucleus. Moreover, computationalmolecular modeling and biochemical studies suggest that, un-like the wild-type protein, this mutant is unable to self-associ-ate. Therefore, these results suggest the mutation of a carboxy-terminal RGG motif affects ORF57 RNA binding, nucleartrafficking, and multimerization.

MATERIALS AND METHODS

Oligonucleotides, plasmids, and antibodies. Oligonucleotides used in reversetranscription-PCR (RT-PCR) analysis and site-directed mutagenesis are listed inTable 1. To generate pORF57GFP and pORF57-myc, ORF57 cDNA was am-

plified by PCR and cloned into pEGFP-N1 (BD Biosciences, Ltd.) andpcDNA3.1MycHisB (Invitrogen), respectively. pORF57GFP-RGG1, -RGG2,and -Leu mutants were generated using a QuikChange II site-directed mutagen-esis kit (Stratagene). The pORF57GFP-RGG1/2 mutant was subcloned using the5,574-bp fragment from pORF57GFP-RGG1 and the 494-bp fragment frompORF57GFP-RGG2 after digestion with ApaI/BamHI. The ORF47 gene wascloned into pCDNA3.1� (Invitrogen). To generate GST-Aly, the Aly openreading frame (ORF) was PCR amplified and cloned into pGEX4T-1 (GEHealthcare). Antibodies to fibrillarin, C23 (Santa Cruz Biotech), green fluores-cent protein (GFP; BD Biosciences), and GAPDH (glyceraldehyde-3-phosphatedehydrogenase; Sigma) were purchased from the respective suppliers. Unlessstated all antibodies were used at a dilution of 1:1,000 for Western blot analysis.

Cell culture and transfection. HEK293T cells were cultured in Dulbecco’smodified Eagle medium (Invitrogen, Paisley, United Kingdom) supplementedwith 10% fetal calf serum (FCS; Invitrogen), glutamine, and penicillin-strepto-mycin. Plasmid transfections were carried out with Lipofectamine 2000 (Invit-rogen, Paisley, United Kingdom) according to the manufacturer’s instructions.

RNA immunoprecipitation assays. HEK293T cells were cotransfected withORF47 and the ORF57 mutants. After 24 h the cells were washed with ice-coldphosphate-buffered saline (PBS) and UV irradiated (900 mJ/cm2) in aStratalinker. Cells were resuspended in 2mls TAP-lysis buffer (10 mM HEPES,2 mM MgCl2, 10 mM KCl, 0.5% NP-40, 5% glycerol containing protease inhib-itor, and 1 �l of RNaseOUT [Invitrogen]/ml) and lysed on ice for 30 min. ProteinA-agarose beads (50 �l of beads/immunoprecipitation) were washed in PBS,followed by incubation with polyclonal anti-GFP antibody (3 �l/IP) at 4°C for 30min. Then, 50 �l of antibody-bead complex was added to 1 ml of cleared lysateand immunoprecipitated at 4°C with end-over-end mixing for 1 h at 4°C. Thebeads were washed in ice-cold NT2 wash buffer (50 mM Tris-HCl [pH 7.4], 150mM NaCl, 1 mM MgCl2, 0.05% NP-40). The beads were next incubated with 100�l of NT2 buffer (containing 0.1% sodium dodecyl sulfate [SDS] and 0.5 mg ofproteinase K/ml) for 15 min at 55°C. To extract the precipitated RNA, 500 �l ofTRIzol reagent (Invitrogen) and 100 �l of chloroform were added, followed byvigorous shaking and centrifugation at 13,000 rpm for 1 min. Next, 3 volumes ofisopropanol, 1/10 volume of 3 M sodium acetate (pH 5.5), and 20 �g of glycogenwere added to the supernatants, followed by incubation overnight at �20°C toprecipitate RNA. The RNA was pelleted by centrifugation at 15,000 rpm for 30min at 4°C and washed in 80% ethanol. These were again pelleted at 15,000 rpmfor 30 min at 4°C before the pellets were air dried and resuspended in 20 �l ofdiethyl pyrocarbonate in double-distilled H2O plus 0.5 �l of RNaseOUT. RNAwas DNase treated using an Ambion DNase-free kit in accordance with themanufacturer’s instructions, and 10 �l was used in RT reactions set up usinggene-specific primers and SuperScript III reverse transcriptase (Invitrogen) ac-cording to the manufacturer’s instructions. RT-negative samples were set upalongside. Then, 2 �l of cDNA was used in a nested-PCR with ORF47 specificprimers.

Immunofluorescence microscopy and FRAP (fluorescence recovery after pho-tobleaching). HEK293T cells were grown on polylysine-treated coverslips. Cellswere fixed in 4% paraformaldehyde and permeabilized in 1% Triton X-100.Then, the cells were blocked in 1% bovine serum albumin (BSA) made in PBSand incubated at 37°C for 1 h. Primary antibodies, C23 and fibrillarin, werediluted 1:100 in 1% BSA and incubated with the cells for 1 h at 37°C. Texas Redanti-mouse (Vector Laboratories, California) or Alexa Fluor 633 anti-rabbitantibody (Invitrogen) was diluted 1:500 in 1% BSA and incubated with the cellsfor 1 h at 37°C. Coverslips were mounted in Vectorshield mounting medium(Vector Laboratories), and staining was visualized on an Upright LSM 510META Axioplan 2 confocal microscope (Zeiss) using LSM imaging software(Zeiss).

TABLE 1. Primers used in site-directed mutagenesis of the ORF57gene and qRT-PCR analysis of the nuclear export assays

Primer Sequence

57RGG1F.................GGC AGA ACA ACA GCG GCC GGA CAG TGT C57RGG1R ................GAC ACT GTC CGG CCG CTG TTG TTC TGC C57RGG2F.................GCT TAA CTT CGC GGC AGG GCT GCT CTT G57RGG2R ................CAA GAG CAG CCC TGC CGC GAA GTT AAG C57LeuF .....................GCA GAG CGA GGC AGC ACT GAT C57LeuR.....................GAT CAG TGC TGC CTC GCT CTG CORF47F ...................CGG GAT CCA TGG GGA TCT TTG CGC TAT TTORF47R...................CCG CTC GAG TGA CTG ATG GAT AGG GTG CCCGAPDHF.................AGG GTC ATC ATC TCT GCC CCC TCGAPDHR ................TGT GGT CAT GAG TCC TTC CAC GAT

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For FRAP analysis, HEK293T cells were plated on glass-based 33-mm culturedishes and imaged at 24 h posttransfection using an Upright LSM 510 METAAxioplan 2 confocal microscope (Zeiss). Cells were maintained at 37°C and,during imaging, the cell culture medium was exchanged for CO2-independentmedium (Gibco). Fluorescence recovery was measured using the ROI meanmodule of the LSM 510 software.

GST-pulldown and immunoprecipitation assays. Recombinant glutathioneS-transferase (GST) and GST-Aly proteins were expressed and purified fromcrude bacterial lysates by incubation with glutathione-Sepharose 4B affinitybeads according to the manufacturer’s specifications (Pharmacia Biotech). Theprotein-bound beads were then incubated with HEK293T-transfected cell ex-tracts previously lysed with lysis buffer (0.3 M NaCl, 50 mM Tris-HCl [pH 8.0],1% Triton X-100; 0.1% SDS) containing protease inhibitors (AEBSF; Roche)for 16 h at 4°C. The beads were then pelleted, washed four times in lysis buffer,and resuspended in Laemmli buffer, and the precipitated polypeptides wereidentified by immunoblot analysis.

To perform coimmunoprecipitation assays, 293T cells were transfected using 2�g of the appropriate DNAs. After 24 h, the cells were harvested and lysed withlysis buffer (0.3 M NaCl, 1% Triton X-100, 50 mM HEPES buffer [pH 8.0])containing protease inhibitors (leupeptin and phenylmethylsulfonyl fluoride).For each immunoprecipitation, 20 �l of GFP-Trap-affinity beads (Chromotek) orMyc-Affinity-agarose (Sigma) was incubated with each respective cell lysate for3 h at 4°C, as directed by the manufacturers. The beads were then pelleted andwashed, and the precipitated polypeptides were resolved on a 12% SDS–poly-acrylamide gel and analyzed by immunoblotting.

Quantitative RT-PCR. To assess the mRNA export efficiency, HEK293T cellswere cotransfected with ORF47 and ORF57 mutants. After 24 h, RNA wasextracted from the total, nuclear, and cytoplasmic fractions by using TRIzol(Invitrogen) as described by the manufacturer. RNA was DNase treated usingthe Ambion DNase-free kit according to the manufacturer’s instructions, andRNA (1 �g) from each fraction was reverse transcribed using SuperScript II(Invitrogen) according to the manufacturer’s instructions, using oligo(dT) prim-ers (Promega). Next, 10 ng of cDNA was used as a template in SensiMixPlusSYBR quantitative PCRs (Quantace), as recommended by the manufacturer,using a Rotor-Gene Q 5plex HRM platform (Qiagen), with a standard three-stepmelt program (95°C for 15 s, 60°C for 30 s, and 72°C for 20 s). With GAPDH asan internal control mRNA, quantitative analysis was performed using the com-parative CT method as previously described (44).

RESULTS

Site-directed mutagenesis of carboxy-terminal RGG motifeffects ORF57 subnuclear localization. KSHV ORF57 is amultifunctional protein that (i) binds viral intronless mRNAs,(ii) shuttles between the nucleus, nucleolus, and the cytoplasm,and (iii) mediates the nuclear export of intronless viralmRNAs. To investigate whether RNA binding is linked to thesubnuclear trafficking of the ORF57 protein, we analyzed thenuclear localization and trafficking potential of an ORF57 mu-tant protein, which is unable to bind RNA. Previous work hassuggested that deletion of a carboxy-terminal RGG motif af-fects the ability of ORF57 to bind RNA (39). Therefore, site-directed mutagenesis was performed on the carboxy-terminalRGG box (residues 372 to 374), termed 57RGG2. In addition,two additional site-directed mutants were generated as suitablecontrols, the first altered a similar amino-terminal RGG motif(residues 138 to 140), termed 57RGG1, whereas the secondaltered a leucine-rich region (residues 341 to 369), termed57Leu, previously shown to affect RNA binding (39). Mutagen-esis was performed altering the wild-type residues to alanineswithin pORF57-GFP (3) (Fig. 1A). Moreover, a double mu-tant was also produced combining both RGG box mutations.Mutations were performed in pORF57-GFP to allow bothindirect immunofluorescence and live subcellular traffickinganalysis to be performed. Sequence analysis confirmed thealteration of the wild-type residues to alanines. Moreover, todetermine that all of the ORF57 site-directed mutants were

expressed at levels similar to the wild-type ORF57, each mu-tant was transfected into HEK293T cells, and cell lysates as-sayed by Western blot analysis using a GFP-specific antibody.The results show that all mutants were expressed at levelssimilar to that for the wild-type ORF57 protein (Fig. 1B).

To confirm that the 57RGG2 site-directed mutant was un-able to bind RNA, an RNA immunoprecipitation assay wasutilized, which we have previously used to show that wild-typeORF57 protein binds to KSHV intronless mRNAs (3). Toperform the RNA immunoprecipitation assays, HEK293T cellswere cotransfected with a pORF47, in the presence of eitherGFP, wild-type ORF57, or each ORF57 mutant (57RGG1,57Leu, 57RGG2, and 57RGG1/2). After 24 h, the cells wereUV irradiated to cross-link interacting RNA and protein. AGFP-specific polyclonal antibody was then used to pull downORF57-RNA complexes, and the isolated RNA was subjectedto RT-PCR analysis. The results show that wild-type ORF57,57RGG1, and 57Leu all bind and precipitate the ORF47 in-tronless mRNA (Fig. 2). In contrast, both 57RGG2 and57RGG1/2 are unable to bind ORF47 mRNA, indicating thatsite-directed mutation of only two amino acid residues withinthe RGG2 box abrogates the ability of ORF57 to bind intron-less mRNAs (Fig. 2). These results are in general agreementwith previous deletion analysis of ORF57 RGG motifs, al-though the results here show that alanine substitutions of the57Leu region retain the ability to bind RNA, in contrast to amutation that alters the leucine-rich region with the moredisruptive proline residues (39).

Having produced a site-directed ORF57 mutant that wasunable to bind RNA, we next determined whether the inabilityto bind RNA had any effect on ORF57 subcellular localizationand subnuclear trafficking. HEK293T cells were grown onpoly-lysine-treated coverslips and transfected with wild-typeORF57 or ORF57 mutants. After 24 h, the cells were fixed andpermeabilized, and wild-type ORF57 and ORF57 mutantswere visualized directly via GFP fluorescence. In addition,indirect immunofluorescence was used to identify the nucleo-lus using C23-specific antibodies. C23 or nucleolin is a majornucleolar phosphoprotein that influences synthesis and matu-ration of ribosomes and is widely used as a nucleolar marker.The results show that wild-type ORF57, 57RGG1, and 57Leuhave staining similar to that described previously (7), localizingto the nucleolar and nuclear speckles (Fig. 3). However, it mustbe noted that 57RGG1 fluorescence seems more diffusethroughout the nucleus, with nuclear speckle and nucleolarlocalization. Staining of the nucleolus is typified by a halo-likestructure due to an inability of the staining antibody to pene-trate deep into the dense nucleolus. In contrast, both of themutants which affect the RNA binding ability of ORF57,57RGG2 and 57RGG1/2, showed a distinct staining patternthat lacked colocalization with the nucleolar marker C23. Thissuggests that the alteration of the carboxy-terminal RGG motifwhich ablates RNA binding may also affect ORF57 subcellularlocalization, specifically the ability of ORF57 to localize to thenucleolus (Fig. 3A).

It is also evident that the classical halo staining pattern ofC23 is replaced by a nucleolar-necklace-like staining reminis-cent of a disorganized nucleolar structure in the presence ofthese RGG2 mutants. The nucleolar necklace is formed byrRNA transcription sites extending into the nucleoplasm,

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where each bead of the necklace corresponds to the nucleolarfibrillar component (FC) and dense fibrillar component (DFC)and contains rDNA, rRNAs, polymerase I, DNA topoisomer-ase I, nucleolar transcription factor 1 (UBF), and fibrillarin(46). This aberrant localization could be due to 57RGG2 nolonger localizing to the nucleolus or to the fact that 57RGG2still localizes to the nucleolus; however, in their presence C23is expelled into the nucleoplasm. To further investigate thesubcellular localization of 57RGG2, the cells were also stainedwith a second nucleolar marker, fibrillarin (Fig. 3B). The re-sults demonstrate that fibrillarin shows the same pattern ofstaining as C23, suggesting that the nucleolus is being stainedand that 57RGG2 is no longer localizing to the nucleolus.Therefore, these results suggest that mutation of the ORF57carboxy-terminal RGG motif, which affects its ability to bindRNA, also affects the ability of ORF57 to localize to the nu-cleolus and instead the 57RGG2 mutant localizes to distinctnuclear subdomains.

Fluorescence recovery after photobleaching indicates57RGG2 is unable to traffic in the nucleus. We have previously

FIG. 1. Site-directed mutagenesis of putative RNA binding motifs within the ORF57 protein. (A) Schematic representation of the generatedORF57 site-directed mutants. (B) 293T cells were transfected with either pGFP, pORF57GFP, or the ORF57 mutants. After 24 h, the cell lysateswere analyzed by Western blot analysis using GFP and GAPDH-specific antibodies. GAPDH served as a loading control.

FIG. 2. Site-directed mutagenesis of the carboxy-terminal RGG2motif inhibits RNA binding. 293T cells were transfected with pORF47in the absence or presence of either pGFP, pORF57GFP, or theORF57 mutants. After UV cross-linking, RNA immunoprecipitationswere performed using no antibody or a GFP-specific antibody. TotalRNA served as a positive control for the PCR (input).



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shown that ORF57 can shuttle between the nucleus and cyto-plasm and also traffic through the nucleolus (7). Therefore, thefailure of 57RGG2 to localize to the nucleolus may be due toa defect in its trafficking ability. Hence, FRAP was used toinvestigate the mobility of 57RGG2 compared to the wild-typeprotein and other mutants. Using live cell imaging it is possibleto gauge and compare ORF57 and mutant protein traffickingrates by photobleaching an area of the localized protein. The

fluorescence immediately after the bleach was normalized to 0,and the final fluorescence at equilibrium was designated as 1.57RGG1 and 57Leu show a rate of mobility similar to that ofwild-type ORF57, having almost fully recovered after 20 s (Fig.4). 57RGG2 and 57RGG1/2, however, display a severe inabil-ity to traffic with little recovery of the bleached protein.57RGG2 was reproducibly less mobile than wild-type ORF57,giving rise to a detectable bleach zone immediately after the

FIG. 3. Site-directed mutagenesis of the carboxy-terminal RGG2 motif affects subcellular localization of the ORF57 protein. 293T cells weretransfected with either pGFP, pORF57GFP, or the ORF57 mutants. After 24 h, the cells were fixed and permeabilized, and the GFP fluorescencewas analyzed by direct visualization, whereas indirect immunofluorescence was performed using C23-specific antibodies (A) and fibrillarin-specificantibodies (B) to identify the nucleolus.



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bleach (Fig. 4). These results suggest that mutation of thecarboxy-terminal RGG motif has a major effect on the traf-ficking ability of the protein and consequently on its subcellularlocalization.

Mutation of the RGG2 motif does not affect ORF57 bindingto the hTREX protein, Aly, but has a marked effect on theability of ORF57 to export viral intronless mRNA from thenucleus. To further investigate the implications of the inabilityof the 57RGG2 mutant to traffic within the nucleus, each

mutant was assessed for its capability to interact with cellularnuclear export factors and also for its ability to export anintronless KSHV mRNA into the cytoplasm. To assess whateffect the 57RGG2 mutation had upon known protein-proteininteractions required for ORF57-mediated nuclear export, weassessed the potential of each mutant to interact with thecellular protein Aly, which allows the formation of an export-competent ORF57-mediated ribonucleoprotein particle (3).GST-pulldown experiments were performed using recombi-

FIG. 4. FRAP analysis performed on 293T cells transfected with either pORF57GFP or the ORF57 mutants. (A) Recovery was assessed overa 40-s period after a photobleaching period. (B) Fluorescence recovery curves. Data were normalized following the bleaching period so that theinitial postbleaching was set as 0 and the final intensity was set as 1.



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nant GST and GST-Aly bound to GST-affinity beads and in-cubated with GFP-, ORF57-, or ORF57 mutant-transfectedcell lysates. The results demonstrate that wild-type ORF57 andall ORF57 mutants retain the ability to interact with thehTREX protein, Aly (Fig. 5A). However, it appears that theremay be a reduction in the binding affinity between GST-Alyand the 57RGG2 and 57RGG1/2 constructs compared to theother ORF57 constructs. This suggests that although an alter-ation in the subcellular localization and trafficking of the57RGG2 mutant is observed, this does not prevent, but mayreduce the affinity of, direct protein-protein interactions withcellular nuclear export adapter proteins.

To assess the capability of each ORF57 mutant to enhancethe nuclear export of a viral intronless mRNA, HEK293T cellswere transfected with a plasmid expressing the intronlessKSHV ORF47 gene in addition to either GFP, wild-typeORF57, or each ORF57 mutant. After 24 h, RNA was ex-tracted from total and cytoplasmic fractions, and the RNAlevels were assessed using quantitative RT-PCR as previouslydescribed (7). The total RNA levels were assessed to ensuresimilar expression levels of the ORF47 mRNA. Figure 5Bi

shows that the expression levels from total cell fractions aresimilar in the presence of wild-type ORF57 and each ORF57mutant, although there is a slight reduction in the presence of57RGG2. This is not unexpected since ORF57 RNA bindinghas been implicated in enhancing the stability of some viraltranscripts (42). However, there was a significant change in thelevels of ORF47 mRNA in each cytoplasmic fraction (Fig.5Bii). The results demonstrate that the wild-type ORF57 pro-tein can enhance the cytoplasmic accumulation of the intron-less ORF47 mRNA as previously observed (3). Similarly, site-directed mutagenesis of the RGG1 and leucine-rich region hadlimited, if any, effect on ORF57-mediated nuclear export. Incontrast, however, mutation of the RGG2 box alone or inconjunction with RGG1 had a dramatic effect on the cytoplas-mic accumulation of ORF47 mRNA levels (Fig.5 Bii). Thissuggests that the alteration in the subcellular localization, nu-cleolar trafficking, and RNA-binding ability of the 57RGG2mutant has a dramatic effect on ORF57 function.

57RGG2 is unable to homodimerize. ORF57, similar to itshomologues, is thought to have the ability to self-associate.Previous studies have shown that multimerization domains

FIG. 5. Site-directed mutagenesis of the carboxy-terminal RGG2 motif affects the ability of the ORF57 protein to export intronless mRNAsfrom the nucleus. (A) Recombinant GST- and GST-Aly-bound glutathione-agarose beads were incubated with pGFP-, pORF57GFP-, or ORF57mutant-transfected cell lysate. After several washes, the bound proteins were analyzed by Western blotting using a GFP-specific antibody. (B) 293Tcells were transfected with pORF47 in the absence or presence of pORF57GFP or mutant pORF57GFP. At 24 h posttransfection, the total (i)and cytoplasmic (ii) RNA was isolated from the cells, and the relative levels were analyzed by quantitative RT-PCR using GAPDH as a reference.The fold increase was determined by using the ��CT method, and statistical significance was determined using an unpaired Student t test. Datafrom three independent experiments are presented as the fold increase over the GFP control.



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within the ORF57 homologue, ICP27, are contained with thecarboxy terminus (21, 51). Therefore, to assess whether alter-ations within the carboxy-terminal RGG2 motif had any effecton the ability of ORF57 to multimerize, computational molec-ular modeling studies were performed. The KSHV ORF57amino acid sequence was analyzed using the I-TASSER data-base, a three-dimensional structure prediction algorithm. Pre-diction results demonstrated that ORF57 exhibits a mainlyunstructured amino terminus with a packed alpha-helical car-boxy terminus. In addition, it contains an unstructured flapcontaining mainly positively charged residues, which may havea role in stabilizing homodimerization and/or RNA binding.To assess whether the predicted monomer model could un-dergo self-association, the monomer structural prediction wasimported into the Maestro (Schrodinger) prediction algorithm,which assessed possible dimer interfaces. The results demon-strate that ORF57 is most likely to form a head-to-tail con-catemer (Fig. 6A). Moreover, highlighting the position of thecarboxy-terminal RGG motif on both monomers (indicated inred) suggests that this region of the carboxy terminus may havean important role in the molecular interactions between thetwo monomers.

However, a previous report utilizing a deletion mutant en-compassing the RGG2 motif, instead of a site-directed mutantused herein, suggested that deletion of the RGG2 does notaffect homodimerization (39). Therefore, to further investigatethe possible role of the 57RGG2 motif in ORF57 ho-modimerization, coimmunoprecipitations were performed aspreviously described (4, 9, 23, 25). HEK293T cells werecotransfected with pORF57-myc in the presence of either wild-type ORF57 or mutant constructs. After 24 h, cell lysates wereincubated with GFP-Trap-Affinity agarose beads, and precip-itated proteins were identified by immunoblotting with a Myc-specific antibody (Fig. 6Bi ). The results demonstrate thatORF57-myc can interact with wild-type ORF57, 57RGG1, and57Leu. In contrast, 57RGG2 or 57RGG1/2 were unable toform homodimers, suggesting that the mutation of the C-ter-minal RGG2 motif affects ORF57 self-association. To confirmthese results, reciprocal coimmunoprecipitations were per-formed using anti-Myc-Affinity beads and immunoblottingwith a GFP-specific antibody. Moreover, these experimentswere performed in the absence or presence of RNase (Fig. 6Bii). Again, results show that site-directed mutagenesis of theRGG2 motif affects ORF57 multimerization. Furthermore,wild-type ORF57 can still form multimers in the absence ofRNA, suggesting that RNA bridging is not required for mul-timerization.

DISCUSSION

KSHV ORF57 is a multifunctional regulator of gene expres-sion assuming many roles during the course of the KSHV lyticreplication cycle, including transcriptional activation, enhanc-ing viral splicing and viral mRNA accumulation, and viralmRNA translation. Moreover, it has a pivotal role in enhanc-ing the nuclear export of viral intronless mRNAs. Central tothis function is the ability of ORF57 to shuttle between thenucleus and the cytoplasm, bind intronless viral transcripts,traffic through the nucleolus, and recruit cellular mRNA ex-port factors forming an export-competent viral ribonucleopro-

tein particle (6, 35). Although ORF57 has a number of putativeRNA-binding domains, it is unclear what region of ORF57 isresponsible for recognizing mRNA. Two RGG box motifs arefound in ORF57, similar to the motif responsible for bindingviral mRNA in HSV-1 ICP27 (29). In addition, ORF57 con-tains a unique leucine-rich region also implicated in RNAbinding (39). Whether these domains are definitive RNA-bind-ing domains or disrupt ORF57 in other ways that have a sec-ondary effect on RNA binding is unknown.

Furthermore, the implications on subcellular trafficking,hTREX complex recruitment and ultimately nuclear mRNAexport, of an ORF57 protein unable to bind RNA have notbeen fully investigated. Therefore, we have assessed here theseORF57 properties using the 57RGG2 mutant, which we haveshown fails to interact with viral intronless RNA in RNAimmunoprecipitation assays. Interestingly, differences were ob-served regarding the subcellular localization and nuclear traf-ficking ability of the ORF57 protein when we compared thewild-type protein and an ORF57 protein unable to bind RNA.Wild-type ORF57, 57RGG1, and 57Leu all localize to thenuclear speckles and nucleolus as previously described (7, 34);however, the ORF57 RNA-binding mutants 57RGG2 and57RGG1/2 displayed a distinct staining pattern lacking nucleo-lar localization. In addition to localizing to a distinct nuclearsubdomain, 57RGG2 also failed to traffic through the nucleo-lus and appears to disrupt the structure of the nucleolus. In thepresence of 57RGG2, the classical halo structure of the nucleo-lus is replaced by a necklace-like stain reminiscent of a disor-ganized nucleolar structure.

The nucleolar necklace is formed by rRNA transcriptionsites extending into the nucleoplasm, where each bead of thenecklace corresponds to the fibrillar nucleolar component (FCand DFC) and contains rDNA, rRNAs, polymerase I, DNAtopoisomerase I, UBF, and fibrillarin (46). A similar stainingpattern is observed in nucleophosmin/B23-depleted cells (1).Nucleophosmin/B23, like nucleolin/C23, is a nucleolar hubprotein that has been suggested to be an important componentof nucleolar protein-protein interaction networks that may inturn have a structural role in the formation and maintenance ofthis dynamic nuclear organelle (18). This is of particular im-portance since the nucleolus consists of complex protein-pro-tein and protein-nucleic acid interactions and, although it isimaged as a steady-state structure, high-throughput proteomicanalysis of purified nucleoli coupled with live-cell imaging offluorescently labeled nucleolar proteins has revealed a dy-namic nuclear compartment, the components of which un-dergo continuous exchange with the nucleoplasm. Therefore,one possibility for the formation of the nucleolar necklace inthe presence of 57RGG2 is that ORF57 binds and sequestersa nucleolar hub protein that gives rise to the disorganizednucleolar structure observed. The interaction between ORF57and nucleolar hub proteins is now under investigation. Thequestion remains, however, about the specific role of the nu-cleolus in ORF57-mediated nuclear export. A number of dif-ferent RNAs are known to be processed during nucleolar traf-ficking. It is possible that the herpesvirus mRNA is also beingmodified in a similar fashion. Alternatively, nucleolar traffick-ing of viral mRNPs could allow the virus to avoid surveillancemechanisms that may otherwise degrade foreign viral intron-less mRNAs prior to translation. However, these results sug-



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FIG. 6. Site-directed mutagenesis of the carboxy-terminal RGG2 motif affects the ability of the ORF57 protein to self-associate. (A) TheORF57 amino acid sequence was analyzed using the I-TASSER database to predict the monomer structure. The monomer structural predictionwas then imported into Maestro (Schrodinger) and free energy minimized. Possible dimer interfaces were identified manually, and selectedstructures were subjected to molecular dynamics to aid in the identification of possible conformations. A molecular model of the putative ORF57dimer (i) and a 50% transparent space-fill model with ribbons (ii) are shown. The RGG motif is indicated in red. (B) For subpanel i, 293T cellswere transfected with pORF57-myc in the presence of either pGFP, pORF57GFP, or ORF57 mutants. After 24 h, cell lysates were included withGFP-Trap-Affinity beads. Precipitated proteins were detected by Western blot analysis using myc-specific antibody. For panel ii, 293T cells weretransfected with pORF57-myc in the presence of either pGFP, pORF57GFP, or p57RGG2. After 24 h, cell lysates were either mock treated (leftpanel) or RNase treated (right panel) for 30 min and then precipitated with Myc-Affinity beads. Precipitated proteins were detected by Westernblot analysis using GFP-specific antibody.



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gest that ORF57 mutants, which cannot traffic through thisorganelle, are deficient in activity.

The results presented here also suggest that KSHV ORF57has the ability to self-associate. The ability to form multimersis likely to be a conserved property within the herpesvirusORF57 protein family (30, 51). Moreover, like its homologues,ORF57 self-association is probably not dependent on otherfunctional motifs involved in intracellular transport, key pro-tein-protein interactions with cellular nuclear export factors, orRNA binding. However, the 57RGG2 mutant, which is unableto self-associate, also lacked the ability to bind RNA and trafficwithin the nucleus, which are both essential for ORF57-medi-ated nuclear export of viral intronless mRNAs.

Computational molecular modeling suggests that ORF57most likely forms an intermolecular head-to-tail concatemer.At present, our data suggest that a region of the carboxyterminus, encompassing the RGG2 motif, is important for thisconcatemer formation. However, whether the site-directedmutagenesis of this motif affects important molecular interac-tions required for self-association or whether these alterationshave generally affected the folding of the carboxy terminus,preventing self-association, is yet to be elucidated. The con-served zinc-finger-like domain has been shown to be importantin ICP27 self-association, which suggests that the 57RGG2mutation has affected the overall structure of the ORF57 car-boxy terminus. Notably, we assessed the subcellular localiza-tion of additional carboxy-terminal deletions of ORF57 anddemonstrated that they also aggregate in subnuclear structuressimilar to the 57RGG2 mutant, suggesting the alterationswithin the carboxy terminus have a detrimental effect on theORF57 protein (unpublished observations). Alternatively, thecarboxy-terminal RGG2 motif may undergo a posttransla-tional modification such as arginine methylation important forprotein-protein interactions. Interestingly, the ICP27 RGGbox is methylated but, surprisingly, this modification has littleeffect on the RNA-binding properties of ICP27, instead affect-ing protein-protein interactions with SRPK1 and Aly (47).Further work is under way to address these questions.

In summary, these results suggest that site-directed muta-tions of a carboxy-terminal RGG motif has detrimental effectson the ability of the ORF57 protein to bind RNA and to trafficin the nucleus and self-associate, which are essential for theORF57-mediated nuclear export of viral intronless mRNAs.

ACKNOWLEDGMENTS

We thank Gareth Howell, Leeds Bioimaging and Flow CytometryFacility, University of Leeds, for useful advice.

This study was supported in part by BBSRC and Wellcome Trustproject grants and by an MRC studentship to A.W. AW. is the recip-ient of a BBSRC Research Development Fellowship.

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